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. 2011 May 10;6(5):e19672. doi: 10.1371/journal.pone.0019672

Figure 4. Evaluation of chimeric ORF2p expression and functionality.

Figure 4

A. Detection of ORF2 protein. Western blot analysis of extracts from transiently transfected HeLa cells with constructs expressing the following human-mouse ORF2 chimeric proteins: 1- MMmhh, 2- HHhmm, 3- HHM, 4- MMH, 5- HHhmh, 6- HHmhh, and 7- optimized human ORF2 (HHH). An arrow indicates the expected position of the 149 kDa ORF2 protein. B. Evaluation of endonuclease activity. Alkaline comet assay for the detection of DNA breaks of transiently transfected cells plasmids expressing the optimized human ORF2 (HHH), an endonuclease and reverse transcriptase mutant (EN−RT−), and the three chimera that are incapable of inducing retrotransposition above background levels (HHM, HHhmm, and HHhmh). The experiment is one representative of three repetitions. Results from Student paired T-test relative to the empty plasmid control (vector) are indicated. Significant differences were observed for all ORF2 constructs, except for the RT− and EN− ORF2 mutant (negative control). Note that all cells are exposed for the gamma-irradiated control (IR), while only a portion of the cells transfected with the ORF2 constructs express the ORF2 protein of interest due to the transfection efficiency. C. Schematic of the LEAP assay. The LEAP assay was previously developed for the detection of L1 ribonucleoprotein (RNP) complexes containing ORF2p and an RNA template [47]. The assay consists of a partial purification of a cytoplasmic subcellular fraction containing ORF2p and L1 RNA as part of the L1 RNP. Isolated RNPs, along with nucleotides and a linker oligo (to function as the priming site), were used for in vitro detection of the reverse transcriptase activity of the L1 ORF2p [23]. Two types of linkers are used: one with “no anchor” having only thymidine residues at the 3′ end (NA; not shown) and one with two residues to function as an anchor at the 3′ end (NV; shown). The generated cDNA is detected by PCR amplification using a primer complementary to the cDNA and to the sequence of the linker oligo. D. Evaluation of reverse transcriptase activity. Reverse transcriptase activity of the different ORF2 chimera was assessed with the LEAP assay using primers to detect cDNA generated from the ORF2 transcript. Extracts from HeLa cells transfected with the different ORF2 chimeric constructs were assayed using the linker with no anchor (NA) or the linker with anchor (NV). No linker oligo was added to the negative control (−). The empty plasmid (vector) and the RT− and EN− ORF2 mutant were used as negative controls. The arrow indicates the expected size product for a positive result.